The present invention relates to a human cDNA encoding a methionine aminopeptidase type-3 (MetAP-3) protein. The invention also relates to nucleic acid molecules associated with or derived from this cDNA including complements, homologues and fragments thereof, and methods of using these nucleic acid molecules, to generate, for example, polypeptides and fragments thereof. The invention also provides methods of using the nucleic acids, for example, to produce a protein and fragments thereof and to screen for compounds or compositions that preferentially or specifically effect the activity of a MetAP-3 protein.
Angiogenesis, the process of new blood vessel formation, is essential for the exponential growth of solid tumors and tumor metastasis. Radiological and cytocidal treatments, combined with regimens involving selective inhibitors of angiogenesis should lead to dramatic reductions in tumor growth. One angiogenesis inhibitor was first discovered as a fungal contaminant of bovine endothelial cell cultures that inhibited cell proliferation (Ingber et al. Nature 348:555-557, 1990). This product was subsequently isolated from A. fumagatus and identified as fumagillin, a well-known amebicide and antibiotic (McCowen et al., Science 113:202-203 (1951)). Fumagillin was found to be a potent inhibitor of endothelial cell proliferation, but its therapeutic window was insufficient for further clinical advancement. TNP-470, a fumagillin-like derivative with 50-fold higher potency, was subsequently developed from a directed chemical approach (Ingber et al., Nature 348:555-557 (1990), Kusaka et al., Biochem. Biophys. Res. Commun. 174:1070-1076 (1991)). This compound""s therapeutic use is limited, however, by its lack of oral availability and dose-limiting neurotoxicity.
Until recently, the molecular target for fumagillin or TNP-470 was unknown. In 1997, the target protein was isolated, purified, and identified by mass spectrometry as the type-2 methionine aminopeptidase (MetAP-2). Both fumagillin and TNP-470 are now known as potent inhibitors of MetAP-2, but not the type-1 enzyme. This result identified MetAP-2 as an anti-angiogenesis target (Sin et al. Proc. Natl. Acad. Sci. (U.S.A.) 94:6099-6103 (1997), Griffith et al. Chem. Biol. 4:461-471 (1997).
The methionine aminopeptidases were first isolated from eubacteria and shown to be cobalt-containing enzymes with molecular masses of about 30 kDa (Ben-Bassat et al., J. Bacteriol. 169:751-757 (1987), Suh et al., Gene 169:17-23 (1996), and Miller et al., Proc. Natl. Acad. Sci. (U.S.A.) 91:2473-2477, 1987). The structure of these enzymes consists of a novel protease fold with pseudosymmetry around a pair of cobalt ions (Roderick and Matthews, Biochemistry 32:3907-3912, 1993).
Enzymes with the same substrate specificity, but with larger molecular masses, were isolated from yeast and pig. Highly homologous regions at the C-terminal domain (xcx9c30 kDa) of the eukaryotic and the prokaryotic forms were discovered, although the N-terminal domain of the eukaryote enzymes was found to be unique (Kendall and Bradshaw, J. Biol. Chem. 267:20667-20673 (1992)). The N-terminal domain of the yeast enzyme contained sequences consistent with two zinc-finger structures, indicating a potential site of nucleic acid interaction. This class of enzyme was designated methionine peptidase Type I (MetAP-1). The porcine enzyme lacked the zinc-binding domains, but contained a block of polylysine and aspartic residues within the N-terminal domain, and was described as Type II (MetAP-2). Both isozymes have been found from Archebacteria to man, indicating a critical metabolic function (Arfin et al. Proc. Natl. Acad. Sci. (U.S.A.) 92:7714-7718 (1995), Bradshaw et al., TIBS 23: 2.63-267 (1998)).
Methionine aminopeptidase-2 is bi-functional. One action is the removal of the N-terminal methionine residues from their protein substrates. MetAP-2 can also bind to and prevent phosphorylation of the xcex1-subunit of the peptide change initiation factor eIF-2 by one or more eIF-2 kinases (Datta et al., Proc. Natl. Acad. Sci. USA 85: 3324-2238 1(1988), Wu et al., J. Biol. Chem. 268:10796-10781 (1993)). This action promotes protein synthesis within the cell. The eIF-2 phosphorylation inhibitory activity of MetAP-2 is unaffected by TNP-470 binding, indicating that the loss of aminopeptidase activity is involved in the anti-angiogenic activity of TNP-470 (Griffith et al., Chem. Biol. 4:461-471 (1997)). The function of methionine peptidase activity in endothelial cell proliferation during tumorigenesis is unclear, although inhibition of MetAP-2 may play a role in altering the stability of one or more protein(s) whose abnormal presence or absence results in endothelial cell dysregulation. Several signaling proteins also appear to be modified by the covalent attachment of myristic acid to a glycine residue which occurs only after the initial amino-terminal methionine removal by MetAP-2 (Peseckis et al., J. Biol. Chem. 267:5107-5114 (1993)). Inhibition of methionine aminopeptidase activity may prevent this covalent attachment, resulting in improper functioning of a signal component specific to endothelial cell cycle regulation (Sin et al. Proc. Natl. Acad. Sci. (U.S.A.) 94:6099-6103 (1997)).
N-terminal processing agents, such as the methionine aminopeptidases, also function to initiate post-translational peptide or protein modifications which may control or induce activation, translocation, or protein turnover (Bradshaw et al., TIBS 23: 263-267 (1998)). Because this initial processing is important for normal protein functioning, it is possible that alteration of methionine aminopeptidase activity is a factor in a variety of diseases, including angiogenesis. Therapies can thus be developed which can modify methionine aminopeptidase activity to restore proper protein processing.
Methionine aminopeptidase activity can also be used to modify recombinant proteins expressed and harvested from E. coli or other expression systems. Recombinant proteins that retain the N-terminal methionine, in some cases, have biological characteristics that differ from the native species that retain the N-terminal methionine, including the induction of undesireable antibodies. Using a methionine aminopeptidase for recombinant protein modification provides a low-cost method of generating potentially life-saving therapeutic proteins and to mimic the structure of native protein species which are used to combat or eliminate the causes of various diseases (Sandman et al., Biotechnology (N Y) 13:504-6 (1995)).
Clearly, an understanding of methionine aminopeptidase activity and its role in various tissues can provide useful therapeutic and diagnostic insight into angiogenesis and tumor metastasis. The known MetAP-2 inhibitors are not good candidates for clinical use as angiogenesis inhibitors due to their neurotoxic effects. Differential expression of mammalian MetAP-1, MetAP-2, or other unidentified methionine aminopeptidases may partially or totally account for the observed variation in sensitivity of different cell types to inhibition by TNP-470 and other MetAP-2 inhibitors, and thus account for the observed toxicity of these drugs.
One aspect of the present invention is to provide a novel methionine aminopeptidase, MetAP-3, and its nucleic acids, proteins, peptides, fragments, and homologues.
Another aspect of the invention is to provide new and advantageous targets to screen for diagnostic and therapeutic agents and compositions useful for diagnosis or treatment of angiogenesis-related diseases.
The invention provides a substantially-pure nucleic acid comprising a nucleic acid sequence selected from the group consisting of: SEQ NO: 7 or complements thereof; nucleic acid sequences that specifically hybridize to SEQ NO: 7 or complements thereof, especially those that hybridize under stringent conditions; nucleic acid sequences encoding a MetAP-3 protein or fragment thereof, or complement of these nucleic acid sequences; and nucleic acid sequences encoding the amino acid sequence of SEQ NO: 8, or complements thereof.
In one embodiment, the present invention relates to a substantially-pure nucleic acid selected from the group consisting of: a nucleic acid molecule comprising SEQ NO: 7 or its complement, and fragments of either having a length of about 12 to about 650 nucleotides, and a nucleic acid molecule that encodes a protein having a sequence of SEQ NO: 8 or a fragment of any having a length of about 10 to about 215 amino acids.
The present invention also relates to a nucleic acid encoding a fragment of a MetAP-3 protein, wherein the nucleic acid is about 12 to 650 nucleotides in length and has from about 99% to about 70% identity to a fragment of SEQ NO: 7.
In a particularly useful embodiment, a substantially-pure nucleic acid of the invention will specifically hybridize to a nucleic acid molecule encoding MetAP-3 or a complement thereof and fail to specifically hybridize to a nucleic acid molecule encoding MetAP-1, MetAP-2 or a complement of either.
The present invention also provides a substantially-pure MetAP-3 nucleic acid molecule which comprises a nucleic acid sequence that is identical to at least about 12 contiguous nucleotides of SEQ NO: 7 or its complement.
In a further embodiment, the present invention relates to a substantially-pure MetAP-3 protein or fragment thereof encoded by a nucleic acid sequence encoding a protein having an amino acid sequence of SEQ NO: 8 or a fragment of SEQ NO: 8 having a length of about 10 to 215 amino acids.
The present invention further relates to a substantially-pure MetAP-3 protein or fragment thereof comprising at least 10 consecutive amino acids of SEQ NO: 8, wherein the protein possesses a MetAP activity.
In another embodiment, the present invention relates to a transformed cell having a nucleic acid molecule which comprises a structural nucleic acid molecule, wherein said structural nucleic acid molecule encodes a MetAP-3 protein, peptide, or fragment thereof.
In yet another embodiment, the present invention provides a method for determining a level or pattern of MetAP-3 expressed in a cell comprising: (A) incubating, under conditions permitting nucleic acid hybridization, a marker nucleic acid molecule, the marker nucleic molecule capable of specifically hybridizing to a nucleic acid, molecule that encodes MetAP-3 or complement thereof under high stringency conditions and the marker nucleic acid molecule incapable of specifically hybridizing to a nucleic acid molecule that encodes MetAP-1 or MetAP-2 complements of either under high stringency conditions, with a nucleic acid molecule derived from or within the cell; (B) permitting hybridization between the marker nucleic acid molecule and the complementary nucleic acid molecule derived from or within the cell; and (C) detecting the level or pattern of the hybridization. The level or pattern of the hybridized complementary nucleic acid is predictive of the level or pattern of the MetAP-3 protein.
The present invention also relates to a method for detecting the presence of a mutation affecting the level or pattern of MetAP-3 expression comprising the steps: (A) incubating, under conditions permitting nucleic acid hybridization, a marker nucleic acid molecule, the marker nucleic acid molecule comprising a nucleic acid molecule that is linked to a gene, the gene specifically hybridizes to a nucleic acid molecule having a nucleic acid sequence of SEQ NO: 8 or the complement thereof, with a nucleic acid molecule derived from or within said cell, wherein hybridization between the marker nucleic acid molecule and the nucleic acid molecule derived from or within the cell permits the detection of a polymorphism whose presence is predictive of a mutation affecting the level or pattern of the MetAP-3 protein in the cell; (B) permitting hybridization between the marker nucleic acid molecule and the nucleic acid molecule derived from or within the cell; and (C) detecting the presence of the hybridization.
In another embodiment, the present invention provides a method for detecting the presence or absence of angiogenic activity in a mammal which comprises assaying the concentration of a molecule whose concentration is dependent upon the expression of a MetAP-3 gene, the molecule being present in a sample of cells or bodily fluid of said mammal, and comparing the concentration of that molecule in the angiogenesis model animal with the concentration of the molecule in a sample of cells or bodily fluid of a control mammal.
In a further embodiment, the present invention relates to a prognostic or diagnostic method for identifying angiogenesis of a tumor in a patient which comprises the steps: (A) incubating, under conditions permitting nucleic acid hybridization, a marker nucleic acid molecule, said marker nucleic acid molecule comprising a nucleotide sequence that specifically hybridizes to a polynucleotide that is linked to a MetAP-3 gene, with a nucleic acid molecule derived from or within a cell or a bodily fluid of said patient, wherein nucleic acid hybridization between said marker nucleic acid molecule and said nucleic acid molecule derived from or within a cell or bodily fluid of said patient is capable of detecting a polymorphism whose presence is predictive of a mutation affecting MetAP-3 response in said patient; (B) permitting hybridization between said marker nucleic acid molecule and said nucleic acid molecule derived from or within a cell or bodily fluid of said patient; and (C) detecting the presence hybridization.
In another embodiment, the present invention relates to a method of determining an association between a polymorphism and a trait comprising: (A) hybridizing a nucleic acid molecule specific for the polymorphism to genetic material of a cell, wherein said nucleic acid molecule comprises a nucleotide sequence of SEQ NO: 5 or complements thereof; and (B) calculating the degree of association between the polymorphism and the trait.
The present invention also relates to a method of producing a cell capable of overexpressing a MetAP-3 protein comprising: (A) introducing into a cell with a functional nucleic acid molecule, comprising a nucleic acid sequence of SEQ NO: 5, and (B) culturing the cell. The invention also provides a cell and progeny of a cell produced by such a method.
In another embodiment, the present invention relates to a method for detecting a modification in the methionine-removal activity of cells in a mammal, comprising assaying the concentration of a molecule whose concentration is dependent upon the expression of a MetAP-3 protein, the molecule being present in a sample of cells or bodily fluid of the mammal, and comparing to the concentration of that molecule with that in a sample of cells or bodily fluid from a control mammal.
The present invention also relates to a composition comprising an oligodeoxynucleotide and a pharmaceutically acceptable carrier, the oligodeoxynucleotide comprising a sequence set forth in one of: SEQ NO: 7 or its complements or fragment of either, having a length of about 12 to about 650 nucleotides.
The present invention further relates to a composition comprising a polypeptide and a pharmaceutically acceptable carrier, said polypeptide comprising an amino acid sequence encoded by a nucleic acid comprising SEQ NO: 7, or its complement or a fragment of either, having a length of about 12 to about 650 nucleotides; a nucleic acid encoding a protein having a sequence of SEQ NO: 8 or a fragment of SEQ NO: 8 having a length of about 10 to about 215 amino acids.
In another embodiment, the present invention provides a method of producing a cell capable of expressing reduced levels of a MetAP-3 protein comprising: (A) introducing into a cell a functional nucleic acid molecule, comprising a nucleic acid sequence of SEQ NO: 7, wherein the functional nucleic acid molecule results in co-suppression of the MetAP protein; and (B) culturing the cell. The invention also provides a cell and progeny of a cell produced by such a method.
In a further embodiment, the present invention provides a method for reducing expression of a MetAP-3 protein in a cell comprising: (A) introducing into a cell with a nucleic acid molecule, said nucleic acid molecule having an exogenous promoter region which functions in a cell to cause the production of a mRNA molecule, wherein said exogenous promoter region is linked to a transcribed nucleic acid molecule having a transcribed strand and a non-transcribed strand, wherein the transcribed strand is complementary to a nucleic acid molecule having a nucleic acid sequence of SEQ NO: 7 or its complement and said transcribed strand is complementary to an endogenous mRNA molecule; and (B) culturing said cell. The invention also provides a cell and progeny of a cell produced by such a method.
The present invention also provides a method for detecting a compound or composition that modifies the protein phosphorylation activity of a MetAP-3 protein, or a fragment or fusion thereof comprising contacting the compound or composition with a MetAP-3 protein in the presence of a phosphorylating activity and a substrate, allowing a phosphorylation to occur, and detecting the phosphorylation of the substrate in comparison to a control.
The invention also provides a method of isolating a nucleic acid that encodes a MetAP-3 protein or fragment thereof comprising: (A) incubating, under conditions permitting hybridization, a first nucleic acid molecule comprising SEQ NO: 5, or the complement thereof, with a second nucleic acid molecule obtained or derived from a cell; (B) permitting hybridization between said first nucleic acid molecule and said second nucleic acid molecule; and (C) isolating said second nucleic acid molecule. The invention also provides a cell and progeny of a cell produced by such a method.
In a further embodiment, the present invention provides a method for identifying a molecule, compound, or composition that effects the MetAP activity of a MetAP-3 protein, comprising providing a MetAP-3 protein, contacting the MetAP-3 protein with a test sample comprising a molecule, compound, or composition, and comparing the MetAP activity with a control.
The invention further provides a method of using a, MetAP-3 protein or fragment thereof in an assay for screening test substances for the ability to modulate or maintain an activity possessed by a MetAP-3 protein, comprising contacting a MetAP-3 protein or fragment with a test substance, and determining the presence or level of MetAP-3 activity compared to a control.
FIG. 1 displays phylogenetic trees showing the genetic relatedness of various methionine aminopeptidases
FIG. 1 shows the phylogenetic relationship displayed as a tree diagram, between the amino acid sequences of various methionine aminopeptidases, including MetAP types 1, 2, and 3, and the relationship between full-length human MetAP-3, including the partial amino acid sequence encoded by the clone designated MAP-3 insertion (see text for details).
FIG. 2 shows multiple amino acid sequence alignments of various methionine aminopeptidases
FIG. 2 shows a pairwise alignment between two clones encoding MetAP-3, designated map3gt and GT, to show the position of the 50 base pair insert present in the clone GT, later designated MAP-3 insertion (see text for details).
FIG. 3 shows a restriction map of the plasmid pMON57801
FIG. 3 shows a restriction map of the baculovirus donor plasmid pMON57801, containing the MetAP-1 coding sequence under the control of the Autographa californica nuclear polyhedrosis virus (AcNPV) polyhedrin promoter (pPolh). The expression cassette containing a gentamycin resistance marker, nPolh, a His(6) tag, MetAP-1, and an SV40 poly-A termination signal is flanked by the short arms of the bacterial transposon Tn7. The mini-Tn7 cassette is transposed to an attTn7 attachment site which resides on baculovirus shuttle vector (bacmid) harbored in E. coli cells. Composite bacmids containing the min-Tn7 expression cassette are isolated from bacteria and transfected into insect cells to generate pure stocks of recombinant baculoviruses.
FIG. 4 shows a restriction map of the plasmid pMON57800
FIG. 4 shows a restriction map of the baculovirus donor plasmid pMON57800, containing the MetAP-1 coding sequence under the control of the AcNPV polyhedrin promoter.
FIG. 5 shows a restriction map of the plasmid pMON57503
FIG. 5 shows a restriction map of the baculovirus donor plasmid pMON56503, containing the MetAP-3 coding sequence fused to a His(6) tag under the control of the AcNPV polyhedrin promoter.
FIG. 6 shows a restriction map of the plasmid pMON56502
FIG. 6 shows a restriction map of the baculovirus donor plasmid pMON56502, containing the MetAP3 coding sequence under the control of the AcNPV polyhedrin promoter.
FIG. 7 shows a restriction map of the plasmid pMON56500
FIG. 7 shows a restriction map of the baculovirus donor plasmid pMON56500, containing the MetAP-2 coding sequence under the control of the AcNPV polyhedrin promoter.
FIG. 8 shows transcriptional profiling data comparing expression of cyclophilin, MetAP-1, MetAP-2, and MetAP-3 across various tissue samples
FIG. 8 shows relative expression of cyclophilin, MetAP-1, MetAP-2, and MetAP-3, graphically displayed in FIGS. 9-11, across a library of tissue samples (See Example 4 for details).
FIG. 9 shows the distribution of MetAP-1 transcripts across various tissues
FIG. 9 graphically displays the relative expression of MetAP-1 across a library of tissue samples.
FIG. 10 shows the distribution of MetAP-2 transcripts across various tissues
FIG. 10 graphically displays the relative expression of MetAP-2 across a library of tissue samples.
FIG. 11 shows the distribution of MetAP-3 transcripts across various tissues
FIG. 11 graphically displays the relative expression of MetAP-3 across a library of tissue samples.
FIGS. 12-14 show transcriptional profiling data comparing expression of cyclophilin, MetAP-1, MetAP-2, and MetAP-3 across various tissue samples
FIG. 15 shows transcriptional profiling data comparing expression of cyclophilin, MetAP-1, MetAP-2, and MetAP-3 across various brain samples
FIG. 15 shows relative expression of cyclophilin, MetAP-1, MetAP-2, and MetAP-3, graphically displayed in FIGS. 16-18, across a library of tissue samples (See Example 4 for details).
FIG. 16 shows the distribution of MetAP-3 in human brain tissues
FIG. 16 graphically displays the relative expression of MetAP-3 across a library of brain tissue samples.
FIG. 17 shows the distribution of MetAP-2 in human brain tissues
FIG. 17 graphically displays the relative expression of MetAP2 across a library of brain tissue samples.
FIG. 18 shows the distribution of MetAP-1 in human brain tissues
FIG. 18 graphically displays the relative expression of MetAP-1 across a library of brain tissue samples.